The goal of this research is to define the mechanism by which the herpes simplex virus ICP8 DNA-binding protein stimulates late viral gene expression. This viral gene product plays a dual role in viral replication by promoting viral DNA synthesis and independently stimulating late gene expression. Our long term goals are to understand how the utilization of ICP8 for two different functions at two different sites within the cell nucleus is regulated. These proposed studies are of medical relevance because if an antiviral agent could block both functions of ICP8, it would provide a double block to viral infection. In addition, these studies are of general interest in that ICP8 provides a system to study how a ssDNA- binding protein stimulates gene expression. From the study of a trans- dominant mutant form of ICP8, it has been concluded that the mutant form of ICP8 inhibits a normal function of ICP8 in promoting viral late gene expression. The effect of ICP8 on late gen expression will be determined to be transcriptional or post-transcriptional by analysis of rates of transcription of late genes in the presence or absence of the mutant gene product. The mechanism of trans-dominance will be studied by determining which functions of ICP8 such as nuclear localization and DNA-binding are required for this effect. If the effect of ICP8 on late gene expression is transcriptional, the structure of viral chromatin will be examined to determine whether the mutant form of ICP8 is bound to viral DNA when it blocks late gene expression, whether the structure of viral chromatin is altered in the presence of the mutant ICP8 molecule by studies involving in vivo footprinting of chromatin over viral late promoter sequences, and whether viral chromatin over late promoters shows single-stranded character and whether this differs in the presence or absence of the mutant gene product. Mutant viruses resistant to the trans-dominant effects of the mutant ICP8 will be isolated in a genetic approach to defining the mechanism of ICP8 stimulation of late gene expression and possibly identifying viral proteins that interact with ICP8. The mechanism of ICP8 transactivation of gene expression in co-transfected cells will be examined to define possible mechanisms of stimulation of gene expression by ICP8. If the effect of ICP8 is post-transcriptional, viral late RNA processing and the structure of ribonucleoprotein complexes will be studied.